Cell targeting with synthetic sense-and-respond protease circuits
Abstract
A fundamental challenge in biomedicine is developing therapeutics that can target specific cell populations.
Synthetic protein circuits, based on engineered proteins that interact with one another and with endogenous
cellular pathways, could provide a powerful platform to address this challenge. These circuits could directly
sense key cellular pathways, process that information to classify the cellular state, and respond by
conditionally triggering cell death or other beneficial responses. Synthetic protein circuits could also be
encoded as mRNA and delivered transiently to avoid genome modification. We recently showed that viral
proteases could be engineered to regulate one another in a modular way, allowing construction of diverse
protein-level functions from a limited number of components. However, key challenges remain: Can
engineered protease circuits be generalized to sense multiple cancer pathways with minimal perturbations to
the cell? Can they implement a broader set of signal processing capabilities, including thresholding,
integration, and dosage compensation to allow for versatile and precise function in diverse cell contexts? And,
can they selectively target cancer cells when transiently delivered as mRNA? Here, we aim to develop this
system into a broader platform for targeting cancer cells by creating new pathway sensing capabilities,
designing flexible signal processing modules, and demonstrating the ability to sense and kill specific target cell
types. We will focus on cellular models of hepatocellular carcinoma (HCC), a disease which remains
challenging to treat but is relatively permissive for mRNA delivery. In Aim 1, we will design and validate
protease sensors of major oncogenic pathways that play critical roles in HCC. These sensors conditionally
activate viral proteases in response to the localization, clustering, activity, or abundance of target proteins. In
Aim 2, we will create protease-based circuit modules that actively process these signals. We will engineer
thresholding modules to suppress undesired responses to basal pathway activities in normal cells, and
combinatorial logic modules to allow AND-like integration of signals from distinct sensors. In addition, we will
design dosage compensation modules that make protein expression insensitive to circuit delivery, In Aim 3, we
will design mRNA-delivered circuits that selectively kill HCC cell lines with minimal impact on normal
hepatocytes. This research program will establish the end-to-end feasibility of mRNA-delivered protease
circuits and provide the foundations for future programmable circuit-based therapeutics.